Using DNA aptamers and quantum dots for the detection of proteins or other targets

ABSTRACT

The solutions provided here use DNA aptamers and quantum dots for the detection of bacteria, viruses, proteins or other targets. An example of a method described here comprises: providing a complex of DNA complementary strands, one strand being an aptamer, having one strand covalently linked to a quantum dot, and having the other strand linked to a quencher; and contacting said complex of DNA complementary strands with a microorganism or components thereof, under conditions that permit binding of said aptamer with said microorganism or components thereof. In some examples described here, the methods and systems are extremely simple to use and appear to have several advantages over the traditional ELISA. Since no blocking steps are required and the number of washing steps is reduced, the time required to conduct the test is greatly reduced. In some examples described here, a quantum dot aptamer complex comprises one strand of a duplex DNA molecule linked to the quantum dot by an amide bond. It does not matter if the aptamer or the complimentary strand is attached. However, the strand that is not attached contains a non-radiative quencher. Upon addition of the aptamers&#39; target, the amount of light emitted by the quantum dots increases. In some examples described here, the methods and systems can also be used in reverse, with the aptamers&#39; target immobilized on a microtiter plate. This permits an assay like a competitive immuno-assay.

RELATED APPLICATIONS, AND RIGHTS OF THE GOVERNMENT

This application claims the benefit under 35 U.S.C. §119(e) ofprovisional Patent Application Ser. No. 60/959,251, filed on Jul. 12,2007, the entire text of which is incorporated herein by reference. Thisapplication is related to U.S. patent application Ser. No. 11/965,039,entitled Methods and Compositions for Processes of Rapid Selection andProduction of Nucleic Acid Aptamers, filed by Kiel et al. on Dec. 27,2007 (the entire text of which is incorporated herein by reference)which claims the benefit under 35 U.S.C. §119(e) of provisional PatentApplication No. 60/882,454, filed on Dec. 28, 2006. This application isrelated to U.S. patent application Ser. No.12/072,758, entitledAptamer-Based Assays, filed by Jeevalatha Vivekananda and Johnathan L.Kiel on Feb. 27, 2008 (the entire text of which is incorporated hereinby reference), which claims the benefit under 35 U.S.C. §119(e) ofprovisional Patent Application Ser. No. 60/904,900, filed on Mar. 1,2007. The invention described herein may be manufactured and used by orfor the Government of the United States for all governmental purposeswithout the payment of any royalty.

BACKGROUND OF THE INVENTION

The invention relates to assays and more particularly to testingbiological samples.

Conventional immunoassays usually are of the sandwich/capture assay typerequiring a capture antibody or anti-ligand and an identificationantibody with either an enzyme or a fluorescent tag indicating presenceof the ligand of interest.

What is needed is a test that requires fewer steps and less time toconduct.

SUMMARY OF THE INVENTION

The solutions provided here use DNA aptamers and quantum dots for thedetection of bacteria, viruses, proteins or other targets. An example ofa method described here comprises: providing a complex of DNAcomplementary strands, one strand being an aptamer, having one strandcovalently linked to a quantum dot, and having the other strand linkedto a quencher; and contacting said complex of DNA complementary strandswith a microorganism or components thereof, under conditions that permitbinding of said aptamer with said microorganism or components thereof.In some examples described here, the methods and systems are extremelysimple to use and appear to have several advantages over the traditionalELISA. Since no blocking steps are required and the number of washingsteps is reduced, the time required to conduct the test is greatlyreduced. In some examples described here, a quantum dot aptamer complexcomprises one strand of a duplex DNA molecule linked to the quantum dotby an amide bond. It does not matter if the aptamer or the complimentarystrand is attached. However, the strand that is not attached contains anon-radiative quencher. Upon addition of the aptamers' target, theamount of light emitted by the quantum dots increases. In some examplesdescribed here, the methods and systems can also be used in reverse,with the aptamers' target immobilized on a microtiter plate. Thispermits an assay like a competitive immuno-assay.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a simplified diagram showing an example of DNA aptamers linkedto quantum dots, quenching, and dequenching.

FIG. 2 is a simplified diagram showing examples of assays.

FIG. 3 is a graph providing data concerning Example 1.

FIG. 4 is a graph providing data concerning Example 2.

DETAILED DESCRIPTION

We describe examples using aptamers for capturing and reporting thepresence of a target, such as a pathogen. Aptamers are single-strandedoligonucleotides with a length of tens of nucleotides, exhibiting highaffinity and specificity towards any given target molecule. Aptamershave highly defined tertiary structures, which allow them to form stableand specific complexes with a range of different targets, includingamino acids, proteins and whole viruses. In contrast with a conventionalimmunoassay, the example assays described here use DNA aptamers insteadof antibodies in an immunoassay-like procedure. The example assaysdescribed here do not require the formation of a sandwich, and bindingof the ligand of interest causes an increase in signal from thefluorescent marker.

Referring first to FIG. 1, this example comprises a complex of DNAcomplementary strands (duplex 114) covalently linked to a fluorescentnanocrystal (quantum dot 110), and a fluorescent quencher 111.Optionally, a magnetic particle (not shown) may be included in thecomplex. The nanocrystal (q. dot 110) and quencher 111 are on separateDNA strands. In FIG. 1, one strand 113 of a duplex DNA molecule islinked to the quantum dot 110 by an amide bond. It does not matter ifthe aptamer 113 or the complimentary strand 112 is attached to quantumdot 110. However, the strand that is not attached contains anon-radiative quencher 111. Black Hole Quencher 2 (BHQ2) was the kindused in examples described below, but other kinds may be used. Uponaddition of the aptamers' target 120, aptamer 134 and complementarystrand 132 are separated by binding of the target to aptamer 134.Quencher 131 on complementary strand 132 is separated from quantum dot130. The nanocrystal fluorescence is de-quenched and observable by eyeor by a fluorescent reader (fluorometer). The amount of light emitted bythe quantum dot 130 increases (compare well photo 101 and well photo102).

Another example described below (see left side of FIG. 2) comprises acomplex of aptamer duplex 214 covalently linked to fluorescentnanocrystal (quantum dot 210), and a fluorescent quencher, all attachedto the bottom of the well 201 of a microtiter plate. In an alternativeformat, a magnetic particle (micron-sized or a nanoparticle, not shown)is also used to attach the complex to the bottom of the wells of theplate by a magnet placed under the plate. The quantum dot 210 andquencher 211 are on separate DNA strands, complimentary strand 232 andaptamer 213. When these are separated by binding of a target 220, whichthe aptamer 213 is made specifically to bind, the nanocrystalfluorescence is de-quenched and observable by a fluorescent reader(microtiter plate reading fluorometer). In an alternative format, amagnetic particle (not shown) facilitates the separation of the twostrands by magnetic capture of one of them, being attached only to oneof them either covalently by conjugation chemistry or by a DNA positiveand negative strand complementation different from that of the aptamerdouble strand being separated. This complementation of the magneticparticle DNA may be made covalent by chemically cross-linking the twocomplementary strands. The de-quenched complex is either magnetically orcovalently immobilized on the bottom of the wells of the microtiterplate so that the supernatant can be removed or washed away containingthe freed quencher strand of DNA.

The method and system can also be used in reverse (see the right side ofFIG. 2) with the aptamers' target 221 immobilized on a microtiter platewell 202. Aptamer 214 and complementary strand 233 are separated bybinding of the target to aptamer 214. Quencher 212 on complementarystrand 233 is separated from quantum dot 211.

FIG. 1 and FIG. 2 are simplified diagrams showing examples with twoaptamers per quantum dot. However, the invention is not so limited.Example 2 described below has an 8:1 ratio of aptamers to quantum dots,and other ratios may be used.

EXAMPLE 1

Preparation of a Reactive Plate

A maleic anhydride plate (Pierce Biotechnologies) was reacted with anamino dPEG24 acid (polyethylene glycol linker) to provide a tether tothe surface of the plate. This plate was allowed to react overnight incarbonate buffer on the Jitterbug plate shaker. The contents of eachwell were discarded and washed twice with 200 μl of PBS pH 7.0 buffer,then once with 200 μl of methanol. Next, the carboxylic acid end of thistether was then reacted with NHS (N-hydroxy succinimide) and EDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) to coupleNHS to the carboxylic group and set it up for reaction with a primaryamine. The reaction was carried out in methanol. The plate was washedtwice with methanol and resuspended in methanol. The plate was coveredin parafilm and stored in the fridge.

Preparation of Annealed Aptamer Complex

The aptamer strand of approximately 40 nucleotides was annealed to itscompliment which was approximately 21 nucleotides. Either the aptamercontained a 5′ amine and the complimentary a 3′ quencher or the aptamercontained a 3′ quencher and the complimentary strand a 5′ amine. Furthera 3′ amine could be used with a 5′ quencher. The strands were annealedin 10 mM NaCl, 0.1 M MOPS buffer, pH 7.0 by heating to 85° C. forfifteen minutes in a water bath and while still in the water bath cooledto room temperature. Strands were stored refrigerated.

Quenching of Quantum Dots and Immobilization onto Plate

The annealed strands were conjugated to T1 or T2 Carboxyl Birch Yellowquantum dots. These dots and strands were mixed with a molar ratio ofapproximately 8 duplex DNA strands per dot. The reaction was carried outin 0.1 M MOPS, pH 7.0 buffer supplemented to a concentration of 10 mMNaCl. 5mg of EDC was dissolved in 5 ml of MOPS buffer, and 1 ml added tothe reaction. This was repeated three to five times (usually a total of5 mg of EDC was added). The dots were allowed to react overnight and EDA(˜16 mg) was added in the morning along with additional amounts of EDC(3 mg) to couple the EDA. The dots were filtered (Amicon Ultra Spinfilters, 100,000 NMCO) and washed with PBS and Tween 20 (0.025%). Thedots were washed enough to remove all the unreacted EDA. These dots werethen added (50 μl) to a plate with NHS activated carboxyl groups via theamino dPEG24 acid - tether. The plate was allowed to shake for threehours and then left to stand over night w/o shaking. The next morningthe wells were washed with three times with 200 μl PBS.

Plate Assay

Shiga Toxin, purchased as a lyophilized powder in PBS, was reconstitutedto two milliliters using deionized water. This brought the concentrationof toxin to 0.25 mg/ml.

Ovalbumin, purchased from Sigma-Aldrich was also reconstituted indeionized water to bring the final concentration to 1.0 mg/ml.Increasing milliliter quantities of both Shiga Toxin and Ovalbumin wereadded in triplicates to wells containing immobilized dots. These microliter quantities ranged from three up to one hundred. The total volumeof the wells was brought to 100 μl using PBS. The plate was allowed toshake on the Jitterbug plate shaker for one hour, starting at 25° C. andramping up to 37° C. The contents of each well were discarded and washedtwice with 200 μl PBS. The wells were then reconstituted in 100 μl PBSand read using the Synergy Plate Reader.

For results, see FIG. 3. The control wells to which ovalbumin was addedshowed no increase in fluorescence across the entire range of theexperiment. However, while the increase in fluorescence of theimmobilized aptamer-quantum dot complex was not linear, every well towhich shiga toxin was added showed an increase in fluorescence acrossthe entire range of the experiment (0.10 μg to 25 μg of shiga toxinadded).

The indirect assay measures the interference with the baselinede-quenched fluorescence of adding free complex to the bound controlagent. It can measure antibody in the sera of a patient against theagent (when isolating the agent is not practicable) or can be used tomeasure interference with bound de-quenched fluorescence of the complexwhen soluble antigen activates quenched complexes that are removed withthe supernatant wash out.

This example does not require extensive washing (at most a one stepseparation of the freed quencher strand from the covalently boundcomplex or the magnetically bound complex) and does not require separatecapture and reporter anti-ligands. It is not a typical sandwich assay inwhich a separate capture anti-ligand (like antibody) and a separatereporter anti-ligand (like fluorescent antibody or enzyme-linkedantibody) must be added in separate steps with their accompanyingwashing steps.

Finally, some variations to this example are possible. Although thebound complex added to the microtiter plates may be read in situ foragent or by interference for antibody or competition with control boundagent, it can also be transferred by the release of the magneticallycaptured agent for further analysis by orthogonal methods such as PCRperformed on the DNA from the magnetically captured agent or cultureddirectly off these complexes.

EXAMPLE 2

Annealing the aptamer.

Plus STJ-9 was an anti-shiga toxin aptamer, disclosed as “SEQ ID NO:8”by Jeevalatha Vivekananda and Johnathan L. Kiel, in United States PatentApplication 20040023265 A1, Methods And Compositions For Nucleic AcidLigands Against Shiga Toxin And/Or Shiga-Like Toxin, Feb. 5, 2004 (theentire text of which is incorporated herein by reference). The aptamerwas modified with a 3′ amine for attachment to a quantum dot. NegativeSTJ-9 was an oligonucleotide strand complimentary to the 3′ end ofSTJ-9, the anti-shiga toxin aptamer. At its 5′ end it had a Black HoleQuencher (BHQ2) to quench the quantum dot. Oligonucleotides werepurchased from Biosearch Technologies, Inc.

SEQ ID NO:1. Plus STJ-9: 5′ G GTA ACT AGC ATT CAT TTC CCA CAC CCG TCCCGT CCA TAT 3′ SEQ ID NO:2. Negative STJ-9: 5′ ATA TGG ACG GGA CGG GTG T5′BHQ2

The number of moles of the two strands were compared. The strand withthe largest number of nanomoles was dissolved in 1 ml of 0.1 M MOPS(3-(N-morpholino) propanesulfonic acid), 10 mM NaCl, pH 7.2 and a volumetransferred to the other oligonucleotide such that the Negative STJ-9strand would be in excess. This was to assure complete annealing of theamino labeled positive strand, to have no unannealed Plus STJ-9 presentafter the annealing step. The oligonuleotides were annealed (wrapped inaluminum foil to prevent bleaching of the quencher (BHQ2)) with stirringat 75 to 80° C. for 15 minutes. The solution was cooled in the waterbath to room temperature before refrigeration.

Preparation of Positive Control

Plus STJ-9 was dissolved in 1 ml of MOPS buffer. A volume of plus STJ-9was mixed with carboxyl functionalized Birch Yellow Ti Evitags such thatthe ratio of oligonucleotide to Evitag was 4:1. EDC(1-ethyl-3-93-dimethylaminopropyl carbodiimide hydrochloride) dissolvedin MOPS buffer was added to the mixture: dissolve 5 mg EDC in 5 ml of0.1 M MOPS, pH 7.2, add 1 ml to the reaction, and shake on a rotarystirrer at room temperature. This was repeated approximately every 15 to20 minutes for a total of five additions, after which the reaction wasallowed to proceed at room temperature for another 2 hours. To isolatethe Evitags from the reactants and products, an Amicon Ultra CentrifugalSpin Filter was used with a molecular weight cut off of 100,000 Daltons.The Evitags were washed once with 0.05% Tween 20 in PBS and severaltimes with PBS and concentrated to their initial volume beforerefrigeration.

Preparing the Quenched Quantum Dots

The annealed oligonucleotide was mixed with carboxyl functionalizedBirch Yellow T1 Evitags at a ratio of oligonucleotied to Evitag ofapproximately 7:1. EDC (1-ethyl-3-93-dimethylaminopropyl carbodiimidehydrochloride) dissolved in MOPS buffer was added to the mixture:dissolve 5 mg EDC in 5 ml of MOPS and add 1 ml to the reaction and shakeon a rotary stirrer at room temperature. This was repeated approximatelyevery 15 to 20 minutes for a total of five additions, after which thereaction was allowed to proceed at room temperature for another 2 hours.To isolate the Evitags from the reactants and by-products, an AmiconUltra Centrifugal Spin Filter was used with a molecular weight cut offof 100,000 Daltons. The Evitags were washed several times with PBS andconcentrated to their initial volume before refrigeration.

Plate Assay

Shiga Toxin was coupled to Reacti-Bind Maleic Anhydride Plates (PierceBiotechnologies). Lyophilized shiga toxin powder (Toxin Technologies)was reconstituted in 2 ml of Dl water. Ovalbumin (Sigma Aldrich) wasreconstituted in Dl water to give a molar concentration similar to thatof shiga toxin. A carbonate buffer of pH 9.2 was prepared.

To each well of a 96-well Reacti-Bind Maleic Anhydride Plates (PierceBiotechnologies) was added 50 μl of either shiga toxin or ovalbumin and50 μl of bicarbonate buffer. Both proteins were allowed to react withthe plate for two hours at room temperature. The contents of each wellwere discarded, and the washed twice with PBS, pH 7.4. Quenched BirchYellow quantum dots were then added to wells and allowed to react at 37oC for thirty minutes and at room temperature for thirty minutes withshaking. The wells were then washed three times with 200 μl aliquots ofPBS and reconstituted in 100 μl of PBS. The wells were then read by aSynergy Plate reader system.

For results, see FIG. 4. The data is reported in relative fluorescenceunits. Either Shiga toxin or ovalbumin were immobilized on PierceReacti-bind plates. Fifty μl of protein solution were added to the wells(shiga toxin conc 0.25 mg/ml, total protein 1.0 mg/ml; ovalbumin 0.5mg/ml). Number of reactive sites per well were 110 picomoles. Maximumamount of shiga toxin in a well was approximately 3 micrograms (5×10−11mols). To each well was added the indicated volume of theaptamer-quantum dot complex (concentration of qdots is 4.5nanomoles/ml). 25 μl=0.112 nanomols of qdots. Ratio ofaptamer:qdots=8:1. As the volume of aptamer-quantum dot complexincreased, the fluorescence showed a linear increase from the wells withshiga toxin, indicating the aptamer-quantum dot complex was binding tothe toxin and dequenching. The control, ovalbumin, shows no suchincrease in fluorescence, indicating that the aptamer-quantum dotcomplex was not binding either specifically or non-specifically to theovalbumin or the well.

FITC Labeled Anti-Shiga Toxin Antibody

For a comparison of quantum dot/aptamer complex (not quenched) withfluorescently labeled antibody against shiga toxin and ovalbumin, shigatoxin and ovalbumin were immobilized on Pierce Reacti-bind plates andeither qdot/aptamer complex or antibody was added. The data (not shownhere) demonstrated that the aptamer behaved similarly to the antibody.FITC labeled anti-shiga toxin antibody was purchased from ToxinTechnologies to verify that shiga toxin was bound to the Reacti-BindMaleic Anhydride Plates. For binding reactions with the antibody andpositive control, the wells were first blocked with a blocking solutionmade of 3% dry milk in tris-buffered saline, pH 7.5. After 1 hour at 37C, the wells were washed twice with PBS, and either antibody or positivecontrol added and incubated as described for the quenched Evitags.Afterwards the wells were washed 3 times with PBS and reconstituted in100 ul of PBS. The fluorescence was measured using a Synergy PlateReader.

This example does not require extensive washing (at most a one stepseparation of the freed quencher or complementary metallic nanoparticlestrand from the magnetic capture nanoparticle strand) and does notrequire separate capture and reporter anti-ligands. It is not a typicalsandwich assay in which a separate capture anti-ligand (like antibody)and a separate reporter anti-ligand (like fluorescent antibody orenzyme-linked antibody) must be added in separate steps with theiraccompanying washing steps.

Finally, some variations to this Example 2 are possible. Another versionuses two different types of metallic nanoparticles, one magnetic theother not with different metallic compositions. The nanoparticles ofdifferent metallic composition are chemically linked to the aptamer andcomplementary strands, respectively. They are read by elemental analysisof their light emission spectra using laser induced breakdownspectroscopy. When the two strands are joined, the spectrum contains agiven proportion of the spectral lines of the elemental composition ofboth nanoparticles. When the magnetic one is magnetically trapped andthe other is separated by binding of the ligand or chemical or physicalinteraction, then the laser induced breakdown spectroscopy shows a lossof the other particle's metallic elemental spectral lines.

Some variations may detect and identify bioterrorism or biowarfare agentcontamination of the surfaces of military equipment (including theinterior of aircraft) and personnel. Some variations may determine theviability of such agent on such a surface by measuring the binding ofaptamers to surface ligands of biological agents associated with toxicactivity, infectivity, or pathogenicity by the de-quenching or metallicnanoparticle separation method. This use extends to the action ofenzymes such as DNAase, phospholipase or lipase or protease or thecleavage of some other chemical linkage that could remove the binding offluorescent quencher from the nanocrystal surface by a chemical (acid orbase interaction) or physical action (i.e. detergent that dissolves alipid coating on the nanocrystal releasing the quencher or by heatingthe particle to release it) that may or may be not directly associatedwith the melting of the DNA capture element double strand. In someinstances, it would be associated with its cleavage, including modifiedDNA (addition of peptides or other chemical groups susceptible to suchcleavage) or physical release. Some variations may involve nanoparticleLIBS (laser induced breakdown spectroscopy) tagging and separation. Thisassay can also be used with pre-labeled particles (tagged biologicalmaterial) that when collected in a release (i.e. aerosol collected in animpinger, cyclone, or on a filter) the nanoparticles can be collectedand read with LIBS such that the spectral complementation of theparticles is present (that is, still linked by the DNA complementationor by binding to the target biological particles) or separated or lost(because of destruction of the biological agent linker or the DNAcomplementation). Some variations are suitable for testingdecontamination methods and release of biological agents (for instance,in an air ventilation system of a building) and for measuringdestruction of the released agent (loss of complementation). This caneven be done on the fly by using spark induced breakdown spectroscopy(passing the aerosol through a spark gap and looking at the spectrum ofthe light emission from the spark). Some variations may involve labelingof bacterial spores with separate or combined rare earth metals. Thelinkage of the nanoparticle to the biological agent may be directly fromthe metallic nanoparticle or by plasmid DNA containingaptamer/diazoluminomelanin couplets that chelate the rare earth metalsspecifically to the biological target.

Although the complex added to the surface may be read in situ for agent,it can also be collected and further analyzed with greater sensitivityin a fluorometer or with a LIBS or SIBS device (the latter referring tothe metallic nanoparticle only version). Also, the components: DNA,nanocrystals, in some cases diazoluminomelanin (DALM), and metallicnanoparticles require no refrigeration, can be kept in the dry stateuntil used, and are much more stable than fluorescent organic compoundsand antibody or other peptide-based anti-ligands and require a singleexcitation (the nanocrystals and DALM) wavelength (360 nm to 395 nm) toachieve a broad range of emissions (400 nm to 800 nm), or no excitationwavelength at all oust an electrical discharge—i.e. the LIBS or SIBS formetallic nanoparticles or the DALM/chelated metal complexes). They alsoafford magnetic collection for further analysis by other orthogonalmethods.

In summary, examples provided here use DNA aptamers and quantum dots forthe detection of bacteria, viruses, proteins or other targets. Theexamples provided herein are intended to demonstrate only someembodiments of the invention. Other embodiments may be utilized andstructural changes may be made, without departing from the presentinvention.

1. A method comprising: providing a complex of DNA complementarystrands, one strand being an aptamer, having one strand covalentlylinked to a quantum dot, and having the other strand linked to aquencher; and contacting said complex of DNA complementary strands witha microorganism or components thereof, under conditions that permitbinding of said aptamer with said microorganism or components thereof.2. The method of claim 1, wherein said complex of DNA complementarystrands further comprises at least one metallic particle.
 3. The methodof claim 2, further comprising: separating said complex of DNAcomplementary strands, by magnetic capture of one strand; wherein saidat least one metallic particle is magnetic; and optionally, wherein theother strand of said complex of DNA complementary strands is linked to anon-magnetic metallic particle.
 4. The method of claim 1, furthercomprising: immobilizing said complex of DNA complementary strands on amicrotiter plate.
 5. The method of claim 1, further comprising:immobilizing said aptamer's target on a microtiter plate.
 6. The methodof claim 1, further comprising: detecting whether said aptamer's targetis present on an object's surface.
 7. A method comprising: providing acomplex of DNA complementary strands, one strand being an aptamer,having one strand covalently linked to a quantum dot, and having theother strand linked to a quencher; contacting said complex of DNAcomplementary strands with a sample; and detecting whether saidaptamer's target is present in the sample.
 8. The method of claim 7,wherein said contacting further comprises: adding said sample to amicrotiter plate pre-coated with said complex of DNA complementarystrands.
 9. The method of claim 7, wherein no blocking step is required.10. The method of claim 7, wherein said aptamer comprises SEQ ID NO:1.11. The method of claim 7, wherein said aptamer's target is a bacteria,virus, or protein.
 12. A system comprising: a complex of DNAcomplementary strands, one strand being an aptamer, having one strandcovalently linked to a quantum dot, and having the other strand linkedto a quencher; means for contacting said aptamer with a sample; andmeans for detecting whether said aptamer's target is present in thesample.
 13. The system of claim 12, wherein said complex of DNAcomplementary strands further comprises at least one metallic particle.14. The system of claim 12, wherein said means for contacting furthercomprises a microtiter plate pre-coated with said complex of DNAcomplementary strands.
 15. The system of claim 12, wherein said meansfor detecting further comprises a plate reader.
 16. The system of claim12, wherein said aptamer is linked to said quantum dot.
 17. The systemof claim 12, wherein said aptamer's complementary strand is linked tosaid quantum dot.
 18. A kit comprising: packaged together, a microtiterplate pre-coated with the complex of DNA complementary strands of claim12; and wash buffer.
 19. A kit comprising: packaged together, a complexof DNA complementary strands, one strand being an aptamer, having onestrand covalently linked to a quantum dot, and having the other strandlinked to a quencher; and at least one component selected from: metallicnanoparticles, diazoluminomelanin, or wash buffer.